Various efforts have been made to overcome diseases caused by viral infection. However, no effective treatments have been found to prevent and to cure diseases caused by some viruses such as recent serious world-wide epidemic of human immunodeficiency virus (HIV) and annual epidemic of influenza. Chemotherapeutic treatment with antiviral agents has been used to cope with these diseases caused by viral infections. At present, however, antiviral agents which are markedly effective against systemic infection are very few. Viruses proliferate within living cells and their major metabolic function for multiplication depends on living cells, that is, viruses use the growth ability of host human cells for proliferation after infection. Therefore, most chemical substances that suppress the growth of viruses also affect the host cells and exhibit toxicity. Antiviral agents have some essential problems to be solved due to characteristic features of viral infections in addition to their low selectivity between viruses and cells. Particularly, in patients with acute systemic viral infectious diseases symptoms are usually developed after the virus shows maximum growth in vivo, thus agents which prevent infection and/or suppress the proliferation have been required. One possible method for preventing the viral infection is a vaccination, but it often brings about allergic reactions and various side reactions, furthermore it is substantially ineffective against viral mutation.
The inventors found that iron-binding proteins such as lactoferrin (LF), transferrin and ovotransferrin inhibit the infection and growth of influenza virus and cytomegalo virus (CMV) and accomplished an invention (Japanese Un-examined Patent Publication No. 233619 (1990)). LF secreted in milk is known as an iron-binding protein which possesses antimicrobial activity. Human and bovine LFs have been investigated in detail and their total amino acid sequences have been determined (M. W. Rey et al., Nucleic Acids Res., 18, 5288 (1990) and P. E. Mead et al., ibid., 18, 7167 (1990)). Japanese Un-examined Patent Publication No. 233226 (1989) discloses an antiviral agent containing milk proteins such as LF as effective ingredients, explaining that LF is effective against viruses with and without envelope. Recently, the inventors confirmed the inhibitory effect of iron-binding proteins against infection and growth of HIV and accomplished an invention of HIV infection and growth inhibitor containing LF as an effective ingredient (Japanese Patent Application No. 220635 (1992)).
The characteristic features of iron-binding proteins such as LF against viral infection and proliferation have been gradually elucidated; their practical use as an antiviral agent has been expected. The practical use of these proteins as anti-infective and anti-proliferative agents requires 1 no antigenicity when administered and 2 availability in large quantity. Bovine LF and transferrin, and ovotransferrin of hen's egg can be supplied in large quantities but have drawbacks of antigenicity in human body. On the other hand, human LF has no antigenicity but is unresponsible for demands in large quantities.
Lactoferrin, an iron-binding protein, is well known in exhibiting antimicrobial activity, and antibacterial peptides with a part of the amino acid sequences of LF were recently isolated. EP-0474506 discloses two antibacterial peptides (SEQ. ID. NO.: 1) and (SEQ. ID. NO.: 2) derived from human and bovine LFs as shown below. ##STR2##
An antibacterial peptide having the amino acid sequence was named lactoferricin by Bellamy et al. (W. Bellamy et al., Biochim. Biophys. Acta, 1121, 130-136 (1992)). The antimicrobial activities to Gram negative bacteria, yeasts and fungi have been known, but no antiviral activity has been known.
The inventors investigated the isolation of a peptide having inhibitory activity against viral infection and proliferation from protease hydrolysate of LF which shows most potent inhibiting activity against viral proliferation among the iron-binding proteins disclosed in Japanese Unexamined Patent Publication No. 233619 (1990) to identify the active site of LF. Furthermore, the inventors chemically synthesized LF peptide fragments and studied their inhibitory activity against viral infection and proliferation. They found that a peptide fragment corresponding to 20-37 amino acid residues from N-terminal in human LF and a peptide fragment corresponding to 19-36 amino acid residues from N-terminal in bovine LF are active sites for the suppress of the viral infection and proliferation. Investigations on the synthetic peptides found that 1 loop structure by S--S bond is not essential for the activity, which corresponds to 20 Cys-37 Cys in human LF and 19 Cys-36 Cys in bovine LF, respectively, 2 acetylation of N-terminal of the loop peptides or an elongation of amino acid residue provides favorable effect to the activities, and 3 amidation of C-terminal of the loop peptides or the elongation of amino acid residue provides improved activities.
The antimicrobial activity of LF fragment including the peptide loop has been known (W. Bellamy et al., Biochim. Biophys. Acta 1121, 130-136 (1992) and D. Legrand et al., Biochemistry, 31, 9243-9251 (1992)); however, no inhibitory activity against viral infection and proliferation has been reported.